Figure 8

Spectrum of histone H3 modifications during the cycle of the seminiferous epithelium. (a) Western blot (WB) and Immunoprecipitation (IP) experiments using the aH3T3ph, aPMM and aM4M8 antibodies. Total histones from adult mouse testes were extracted by HCl and analyzed by SDS-PAGE (see Fig. 5b). Portions of this materials were used for IP assays shown in this figure. An asterisk denoted the heavy chains of IgG and an arrowhead the trace of histone H3. (NI) represents non-immune serum used for control IP. For details see text. Full length blots are presented in Supplemental Data III. (b) Detailed representation of the H3T3ph (first row), H3T3K4me₃R8me₂ (second row) and PMM marks (third row) during meiotic divisions. Double immunofluorescence experiments with testis sections from 22 day old mice are shown, using H3S10ph as an indicator of dividing cells. DNA has been stained with TO-PRO3. Scale bars, 5 μm. (c) Newly formed early round spermatids (eRS I and II) from 22 day old mice, stained with the aH3T3ph, aM4M8 and aPMM antibodies. Profiles of late round and elongating spermatids are also shown. DNA has been stained with TO-PRO3. Scale bars, 5 μm. (d) Double immunofluorescence experiments with aPMM and aH3S10ph antibodies. The two marks are detected not only in mitotic spermatogonia (left image, Mitosis), but also in dividing spermatocytes of stage XII tubules (right image, Meiosis). Insets show at higher zoom dividing spermatogonia and spermatocytes carrying the PMM and H3S10ph marks. Scale bars, 20 μm. (e) Sections of adult mouse testis stained with an antibody against SCML2. Confocal images confirm the presence of this protein on the XY body of pachytene spermatocytes (left) and on under-condensed regions of the nucleus in late round and elongating spermatids (right). Scale bars, 20 μm. (f) Frozen sections of adult mouse testis stained with aPMM/anti-H3K9me3; anti-H3K9me3/anti-H3K27me3; and aPMM/anti-H3K9ac. Scale bars, 20 μm. In all specimens, DNA has been stained with TO-PRO 3.